Protein synthesis depends on a complex cellular machine comprised of transfer RNA (tRNA), aminoacyl-tRNA synthetases, initiation, elongation, and release factors, as well as ribosomal RNA (rRNA) and ribosomal proteins. Because translation is an essential cellular process, strong stabilizing selection is expected to influence its components. However, results from the genome sequencing projects show that although the general process of translation is conserved throughout evolution, organisms maintain accurate and efficient protein synthesis in many different ways. In particular, there are unexpected differences within and among organisms in the numbers and types as well as in the sequence and structural features of their tRNAs. Thus there is apparently more resiliency in the transitional apparatus than is typically thought. The proposed experiments are motivated by these observations and are designed to answer three related questions. What are the mechanisms that account for sequence variation among isoacceptors? What are the mechanisms that allow organisms to lose "essential" tRNA genes during evolution? What is the extent of impairment in tRNA function that can be tolerated by the cell? The proposed research uses biochemical and genetic approaches to answer these questions. It will examine (i) the role of the anticodon in tRNA function and evolution; (ii) the mechanisms that compensate for tRNA gene loss; and (iii) the levels of tRNA function that are necessary for cell viability and growth. The results will elucidate elements in tRNAs that dictate their amino acid accepting and donating functions during protein synthesis. In addition, they will elucidate the properties of the translational apparatus that influence these activities of tRNAs. Thus the results will be pertinent to the general problem of the extent to which different phenotypes are expressed from a single genotype due to circumstances in the cell. An understanding of this phenomenon is critical to the interpretation of DNA sequence information and to the development of some strategies that have been proposed for controlling bacterial infections by manipulating the translation machinery.